JP2009219376A - Cytoprotective liquid for medical use - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cytoprotective liquid having long term stability which can sufficiently respond to schedule change of transplantation and to transportation delay caused by a traffic disturbance. <P>SOLUTION: This cytoprotective liquid includes: containing L-glutamic acid, L-aspartic acid and L-tyrosine as a non-essential amino acid; setting the concentration ratio between essential amino acids and non-essential amino acids to 1.3-1.7; setting the concentration of sodium ion to 40-50 mEq/L; and setting hypertonicity or osmotic pressure to 1.2-1.4 compared to that of physiological salt solution. Thus, cytoprotective liquid in which cultured cells of human or animals except human can stably be retained at an unfreezing low temperature for a long period of time is provided. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a cell protection solution used for storing and transporting cells, and more particularly to a cell protection solution for cell transplantation of humans or animals other than humans.

  In ischemic heart diseases including angina pectoris and myocardial infarction, sufficient oxygen is not distributed to the myocardial tissue, and if this state continues for a long time, the myocardial tissue is necrotized. Since adult cardiomyocytes exhibit terminal differentiation and poor self-renewal capacity, myocardial tissue does not regenerate once necrotic. As a result, heart failure occurs due to myocardial infarction and subsequent ventricular remodeling, as well as the failure of pressure overload and its compensatory mechanism.

  Currently, the only way to treat heart failure is to wear a left ventricular assist device or ultimately receive a heart transplant. However, the auxiliary artificial heart is merely a bridge to heart transplantation, and heart transplantation has problems such as donor shortage, rejection and complications.

  Under such circumstances, skeletal myoblast transplantation into myocardial tissue is being studied as a new treatment method. In this treatment, skeletal myoblasts contained in muscles collected from the patient's thighs are cultured and proliferated outside the body and injected into the patient's own myocardial necrotic part to suppress the decline in cardiac function. is there.

  Since skeletal myoblasts are collected from patients themselves, there are no fears of infection due to suppression of rejection and immunity, they do not differentiate into other than muscle cells, and they can be cultured in vitro. is doing.

  The mechanism by which skeletal myoblast transplantation suppresses the decline in cardiac function is not yet clear, but as a general rule, the transplanted skeletal myoblasts form myofibers in the myocardium, and the elasticity of the surrounding normal It may be possible to mechanically assist the movement of the myocardium to prevent further decline in cardiac function, or the transplanted skeletal myoblasts secrete cytokines such as VEGF (vascular endothelial growth factor), which may be due to their effects It has been.

  Skeletal myoblasts used for transplantation are necessary by collecting several g of muscle tissue from the patient's own thigh skeletal muscle by biopsy, culturing the isolated skeletal myoblasts over several weeks after enzyme treatment. Cell number is obtained. If the cells are not transplanted immediately after reaching the target number of cells, the cultured cells will be stored frozen until the day of surgery.

  The cryopreservation solution used at this time contains a frost damage protective agent as a component for the purpose of avoiding the frost damage stress received by the cells. As this cryoprotectant, dimethyl sulfoxide (DMSO) is generally used.

  DMSO functions to prevent damage associated with the formation of intracellular ice crystals through the cell membrane. Specifically, the extracellular water component freezes, and the extracellular NaCl concentration and osmotic pressure increase accordingly, and in the process of dehydration of the cells, there is a function of preventing frost damage due to the intracellular dehydration.

  However, DMSO has been designated as a Class 3 solvent in the “Guidelines for Residual Solvents for Pharmaceuticals” (March 30, 1998, Pharmaceutical Trial No. 307). Must be thawed and further washed to remove the cryoprotectant component.

  However, cells that have undergone the freeze-thaw process are in an unstable state, and if such cells are washed, they are prone to breakage, and there is a problem of cell viability after thawing. There are cases where it cannot be secured.

  In order to solve such problems, the cell protection solution described in Patent Document 1 was invented.

  “Formula I” described in Patent Document 1 is a cell protection solution for storage and transportation without freezing intended for transplantation of skeletal myoblasts, 4.5 g / L glucose 5 g / L human serum albumin, 3.7 g / L sodium bicarbonate, 400 g / L potassium chloride, 200 g / L calcium chloride, 0.195 g / L magnesium sulfate, 0.0209 g phosphorus / L sodium hydrogen phosphate / phosphate It is composed of sodium dihydrogen, 0.000014 g / L iron, 1.11 g / L amino acid, 0.24 g / L multivitamin and is prepared to an osmotic pressure of 358 mOsm / kg.

However, the cell protection solution “Formula I” according to the invention described in Patent Document 1 may not have sufficient storage stability to sufficiently cope with transportation disorder during transportation, postponement of transplantation date, and the like.
US Pat. No. 5,543,316

  Therefore, the present invention is to cultivate cells of humans and animals other than humans to the target cell number for the purpose of cell transplant surgery for the treatment of wounds, and then grow them at a low temperature without freezing. It can be stored and transported from the storage facility to the medical institution where the surgery is performed while maintaining high cell viability at low temperatures, and is inevitable for cryopreserved cells during cell transplantation surgery An object of the present invention is to provide a cell protection solution that can be directly injected into a transplantation site without requiring an operation relating to a certain thawing and frost damage protective agent.

  In view of the above, the present invention has been accomplished by intensively studying the composition of the cell protection solution in order to achieve the above object, and as a result, has found that the above object is achieved under any of the following conditions, and has completed the invention. Preferably, by suppressing the sodium ion concentration of the cell protection solution to 40 to 50 mEq / L and adjusting the osmotic pressure to a physiological saline ratio of 1.2 to 1.4, cell swelling at a low temperature is prevented. Three types of amino acids (L-glutamic acid, L-aspartic acid and L-tyrosin) not contained in the cell protection solution described in Patent Document 1 are added, and the final concentration of amino acids is preferably set to 0. 5-1.5 g / L, concentration ratio of essential amino acids to non-essential amino acids (total grams of essential amino acids in 1 L of cytoprotective solution / total grams of non-essential amino acids in 1 L of cell protective solution) The same shall apply hereinafter.) = 1.3 to 1.7, vitamins to a final concentration of 0.02 to 0.3 g / L, human serum albumin to a final concentration of 0.1 to 10 g / L, etc. Including. In the present specification, numerical values representing mass, weight, concentration, temperature, and ratio are represented by significant figures, and are rounded down to the last digit. Thus, for example, 0.02 g / L includes the range of 0.015 to 0.024 g / L. Moreover, in this specification, low temperature means 1-10 degreeC, Preferably it is 2-8 degreeC. Furthermore, unless otherwise specified, animals include humans.

  The cell protection solution of the present invention does not have to worry about loss during cell thawing caused by freezing of cells, and does not require operations such as washing and removal of the cryoprotectant.

  The present invention is described as follows.

  The cell protection solution of the present invention comprises sugar, serum albumin, iron, sodium ion, potassium ion, calcium ion, magnesium ion, “one or two ions selected from the group consisting of bicarbonate ion and carbonate ion”, “phosphorus 1 to 3 ions selected from the group consisting of oxyhydrogen ion, dihydrogen phosphate ion and phosphate ion ”, at least one amino acid essentially containing an essential amino acid, at least one vitamin and an osmotic pressure regulator. It is composed of an aqueous solution containing it, and has a sodium ion concentration of less than 135 mEq / L, an osmotic pressure of 365 to 435 mOsm / kg, and is essentially free from growth factors.

  As the saccharide, any saccharide that can be recovered by the cell in the glycolysis and metabolized can be used. Preferably, it is a monosaccharide, more preferably one or more hexoses selected from the group consisting of D-glucose, D-fructose, D-galactose and D-mannose, more preferably D-glucose. .

  D-glucose is preferably contained in the cell protection solution at a final concentration of 2.0 to 8.0 g / L, more preferably 4.1 g / L.

  For D-glucose, preferably Japanese Pharmacopoeia glucose or glucose injection solution (generic name) is used. More preferably, Terumo sugar injection 50% (Terumo Corp.) is used.

  As serum albumin, serum albumin suitable for cell culture can be used. Serum albumin derived from the same animal as the animal from which the cells are derived is preferred. For example, in the case of a cell derived from a mammal, serum albumin derived from the same kind of mammal is preferable, and in the case of a cell derived from human, human serum albumin is preferable.

  Human serum albumin is preferably human serum albumin (generic name). More preferred is blood donation albumin 25 “Kaketsuken” (Chemical and Serum Therapy Laboratory) or blood donation albumin-Wf (Benesis).

  Human serum albumin is preferably contained in the cell protection solution at a final concentration of 0.1 to 10 g / L, more preferably 5 g / L.

  Serum albumin is intended to serve as a suspending agent to avoid aggregation of suspended cells, but is not to be construed as limited to this intention.

  As the iron, iron-containing compounds that can be used by cells can be used. Preferably, it is one or more iron-containing compounds selected from the group consisting of iron (III) oxide, iron (III) hydroxide, and complexes or clathrates thereof. More preferably, it is a preparation containing one or more selected from the group consisting of sugar-containing iron oxide (generic name), sideferon (generic name) and chondroitin sulfate / iron colloid (generic name). More preferably, it is Fegin (registered trademark) intravenous injection 40 mg (Nichi-Iko Co., Ltd.) which is a sugar-containing iron oxide injection (formulation name).

  Furthermore, the iron-containing compound is preferably contained in the cell protection solution at a final concentration of 5 to 20 μg / L, more preferably 14 μg / L in terms of iron. Therefore, for example, the intravenous administration of phesin 40 mg contains iron oxide corresponding to 40 mg as iron in 2 mL, so 0.7 μL is used to make the final concentration (as iron) 14 μg / L.

Sodium ions are preferably provided by the sodium salt. A preferred sodium salt is sodium bicarbonate (NaHCO ₃ ).

  Sodium bicarbonate is preferably Japanese Pharmacopoeia Sodium bicarbonate or sodium bicarbonate injection (generic name). More preferably, Meiron (registered trademark) 84 (Otsuka Pharmaceutical Factory) is used.

  Sodium ions are preferably included in the cytoprotective solution at a final concentration of less than 135 mEq / L, more preferably 90 mEq / L or less, more preferably 40-50 mEq / L, and even more preferably 45 mEq / L. .

  Accordingly, the sodium hydrogen carbonate is preferably less than 11.1 g / L, more preferably 7.4 g / L or less, still more preferably 3.3 to 4.2 g / L, and even more preferably 3.7 g / L. In the cell protection solution at a final concentration of Thus, for example, 44.05 mL is used when the final concentration is 3.7 g / L using Meiron 84, which is an 8.4% (1M) aqueous solution.

  In the case of humans, the serum sodium ion concentration is in the normal range of 135 to 145 mEq / L. The intention of lowering the sodium ion concentration is to suppress the inflow of sodium ions from the outside of the cell and the resulting increase in intracellular osmotic pressure, but this is not to be interpreted as being limited to this intention.

  Potassium ions are preferably provided by potassium salts. A preferred potassium salt is potassium chloride (KCl).

  The potassium chloride is preferably Japanese Pharmacopoeia potassium chloride, potassium chloride injection (generic name) or potassium chloride injection kit (generic name). More preferred is a KCL injection 10 mEq kit [Terumo] or a KCL injection 20 mEq kit [Terumo] (Terumo Corp.), which is a potassium chloride injection solution kit (generic name).

  Potassium chloride is preferably contained in the cytoprotective solution at a final concentration of 200 to 600 mg / L, more preferably 302 mg / L. Therefore, for example, in order to achieve a final concentration of 302 mg / L using a KCL injection 10 mEq kit [Terumo] or a KCL injection 20 mEq kit [Terumo], which is a 1M aqueous solution, 4.05 mL is used.

Calcium ions are preferably supplied by calcium salts. A preferred calcium salt is calcium chloride (CaCl ₂ ).

  The calcium chloride is preferably Japanese Pharmacopoeia calcium chloride hydrate, calcium chloride injection (generic name) or calcium chloride kit (generic name), and calcium chloride injection 20mEq syringe [Terumo] (Terumo Corporation) Particularly preferred.

  Calcium chloride is preferably contained in the cytoprotective solution at a final concentration of 50 to 800 mg / L, more preferably 151 mg / L. Therefore, for example, to use a calcium chloride injection 20 mEq syringe [Terumo], which is a 0.5 M aqueous solution, to achieve a final concentration of 151 mg / L, use 2.72 mL.

Magnesium ions are preferably supplied by a magnesium salt. A preferred magnesium salt is magnesium sulfate (MgSO ₄ ).

  Magnesium sulfate is preferably magnesium sulfate hydrate (Pharmacopoeia name), magnesium sulfate injection solution (Pharmacopoeia name) or magnesium sulfate kit (generic name), more preferably magnesium sulfate injection 20 mEq syringe [Terumo]. (Terumo Corporation).

Magnesium sulfate (MgSO ₄ ) is preferably contained in the cell protection solution at a final concentration of 0.1 to 0.5 g / L, more preferably 0.2 g / L. Therefore, for example, in order to achieve a final concentration of 0.2 g / L using a magnesium sulfate injection 20 mEq syringe [Terumo] which is a 0.5 M aqueous solution, 3.32 mL is used.

“One or two ions selected from the group consisting of bicarbonate ions and carbonate ions” are preferably provided by a bicarbonate. A preferred bicarbonate is sodium bicarbonate (NaHCO ₃ ).

  The intention of adding bicarbonate ions or carbonate ions is to constitute a carbonate buffer system, but is not construed as being limited to this intention.

  “1 to 3 ions selected from the group consisting of hydrogen phosphate ions, dihydrogen phosphate ions and phosphate ions” are preferably provided by hydrogen phosphate. A preferred hydrogen phosphate is dipotassium phosphate. The dipotassium phosphate is preferably dipotassium phosphate (generic name), dipotassium phosphate injection (generic name) or dipotassium phosphate injection kit (generic name), more preferably phosphoric acid 2 Potassium injection 20 mEq kit [Terumo] (Terumo Corporation).

Dipotassium phosphate (K ₂ HPO ₄ ) is preferably contained in the cell protection solution at a final concentration of 0.085 to 0.14 g / L, more preferably 0.11 g / L. Therefore, for example, in order to achieve a final concentration of 0.11 g / L using dipotassium phosphate injection 20 mEq kit [Terumo], which is a 0.5 M aqueous solution, 4.05 mL is used.

  Amino acids essentially contain essential amino acids corresponding to the animal from which the cells using the cytoprotective fluid are derived. Preferably, it further comprises at least three non-essential amino acids. Preferably, the essential amino acids are L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-valine, L-histidine, L-glutamic acid, L-asparagine Acids, L-tyrosines and / or their salts. Preferably, the non-essential amino acids include L-glutamic acid, L-aspartic acid and L-tyrosine and / or their salts. More preferably, the non-essential amino acids are L-alanine, L-arginine, L-proline, L-serine, glycine, L-cysteine, L-glutamic acid, L-aspartic acid and L-tyrosine and / or their salts. is there.

  Amino acids as a whole are preferably contained in the cell protection solution at a final concentration of 0.5 to 1.5 g / L, more preferably 1.03 g / L. The concentration ratio between the essential amino acid and the non-essential amino acid is preferably a concentration ratio = 1.3 to 1.7, more preferably a concentration ratio = 1.4.

  The amino acid is preferably used as an aqueous solution containing at least one amino acid. It is preferred to use a mixed amino acid formulation (generic name). It is more preferable to use a high-calorie infusion general amino acid preparation injection (generic name), and it is more preferable to use a high-calorie infusion general amino acid preparation (5-2) injection (generic name). It is more preferable to use Variamin® F injection (Merck Pharmaceutical Co., Ltd.) and / or Moripron® F (Ajinomoto Pharma Co., Ltd.).

  The amino acid is added to the cell protection solution with the intention of a stabilizer for the purpose of imparting nutrition to the cells, but is not limited to this intention.

Vitamins include at least one. Preferably, it is selected from the group consisting of vitamin C, vitamin A, vitamin D, vitamin B ₁ , vitamin B ₂ , vitamin B ₆ , niacin, pantothenic acid, vitamin B ₁₂ , folic acid, biotin and / or salts or derivatives thereof. . More preferably, ascorbic acid (vitamin C), retinol (vitamin A), ergocalciferol (vitamin D), thiamine (vitamin B ₁ ), riboflavin (vitamin B ₂ ), pyridoxine (vitamin B ₆ ), nicotinamide ( Niacin), panthenol (pantothenic acid), cyanocobalamin (vitamin B ₁₂ ), folic acid, biotin and / or a salt or derivative thereof.

  The total amount of vitamins is preferably contained in the cell protection solution at a final concentration of 0.02 to 0.3 g / L, more preferably 0.24 g / L.

Vitamins include vitamin C, vitamin A, vitamin D, vitamin E, vitamin B ₁ , vitamin B ₂ , vitamin B ₆ , niacin, pantothenic acid, vitamin B ₁₂ , folic acid, biotin and / or their salts or derivatives. Although it can add independently, you may mix and use 2 or more. In addition, vitamin C, vitamin A, vitamin D, vitamin E, vitamin B ₁ , vitamin B ₂ , vitamin B ₆ , niacin, pantothenic acid and / or a salt or derivative thereof, a comprehensive vitamin preparation (A), and vitamin B ₁₂ , and may be used separately from the comprehensive vitamin preparation (B) containing folic acid, biotin and / or salts or derivatives thereof, or vitamin C, vitamin A, vitamin D, vitamin E, vitamin B ₁ , vitamin B ₂ A multivitamin preparation (C) containing vitamin B ₆ , niacin, pantothenic acid, vitamin B ₁₂ , folic acid, biotin and / or a salt or derivative thereof may be used.

  The multivitamin preparation (A) is preferably an aqueous solution containing ascorbic acid, retinol, ergocalciferol, thiamine, riboflavin, pyridoxine, nicotinamide, pantothenic acid, tocopherol and / or a salt or derivative thereof, The multivitamin preparation (B) is preferably an aqueous solution containing cyanocobalamin, folic acid, biotin and / or a salt or derivative thereof.

  The multivitamin preparation (A) is more preferably 20 g / L of ascorbic acid, 0.2 g / L of retinoid as retinol, 0.001 g / L of ergocalciferol, and 0.6 g / L of thiamine hydrochloride as thiamine. Final concentrations of 0.72 g / L riboflavin sodium phosphate as riboflavin, 0.8 g / L pyridoxine hydrochloride, 8 g / L nicotinamide, 3 g / L pantothenic acid and 2 g / L tocopherol acetate And the multivitamin preparation (B) is more preferably an aqueous solution containing biotin at a final concentration of 0.012 g / L, folic acid at 0.08 g / L and cyanocobalamin at a final concentration of 0.001 g / L, respectively. It is.

  The multivitamin preparation (A) is more preferably a multivitamin preparation for high calorie infusion (1-2) injection (generic name). V. I. -9 Note (Irom Pharmaceutical Co., Ltd.) is more preferable. The multivitamin preparation (B) is more preferably a multivitamin preparation for high calorie infusion (1-3) injection (generic name). V. I. -3 Note (Irom Pharmaceutical Co., Ltd.) is more preferable.

  The vitamin preparation (C) preferably contains ascorbic acid, retinol, ergocalciferol, thiamine, riboflavin, pyridoxine, nicotinamide, pantothenic acid, tocopherol, cyanocobalamin, folic acid, biotin and / or a salt or derivative thereof. It is an aqueous solution.

  The vitamin preparation (C) is more preferably 20 g / L of ascorbic acid, 0.2 g / L of retinoid as retinol, 0.001 g / L of ergocalciferol, 0.6 g / L of thiamine hydrochloride as thiamine, Sodium riboflavin phosphate as riboflavin 0.72 g / L, pyridoxine hydrochloride 0.8 g / L, nicotinamide amide 8 g / L, panthenolic acid 3 g / L pantothenic acid, tocopherol acetate 2 g / L, biotin 0 0.012 g / L, an aqueous solution containing folic acid at a final concentration of 0.08 g / L and cyanocobalamin at 0.001 g / L.

  The multivitamin preparation (C) is preferably a multivitamin preparation for high calorie infusion (1-4) kit (generic name). V. I. -12 kit (Irom Pharmaceutical Co., Ltd.) is preferred.

  Vitamins are intended to be stabilizers intended to nourish cells, but are not limited to this intention.

  The osmotic pressure adjusting agent is preferably selected from polyhydric alcohols or sugar alcohols. A preferred polyhydric alcohol is propylene glycol, and a preferred sugar alcohol is D-sorbitol. More preferably, it is propylene glycol.

  The osmotic pressure adjusting agent is preferably added in an amount such that the osmotic pressure of the cell protecting solution is a physiological saline ratio of 1.2 to 1.4.

  Propylene glycol or D-sorbitol can be added to the cell protection solution alone, but may be added to the multivitamin preparations (A), (B) and / or (C).

  Propylene glycol is preferably contained in the multivitamin preparation (A), (B) and / or (C) at a concentration of 300 g / L.

  The osmotic pressure regulator is intended to maintain cell viability by making the osmotic pressure of the cell-protecting solution hypertonic, thereby preventing cell swelling at low temperatures due to reduced sodium pump function. However, the present invention is not limited to this intention.

  Furthermore, the water used for preparing the cell protection solution is preferably water for injection (generic name) or distilled water for injection (generic name).

  Furthermore, the cell protection solution is preferably sterile and endotoxin-free.

  Here, the amino acid, vitamin, electrolyte, and the like are compared for the cell protection solution described in Patent Document 1 and one embodiment of the cell protection solution of the present invention.

  Regarding the types of amino acids, the cytoprotective solution described in Patent Document 1 is L-isoleucine, L-leucine, L-lysine (as L-lysine and L-lysine acetate), L-methionine, L-phenylalanine, L- Threonine, L-tryptophan, L-valine, L-histidine, L-alanine, L-arginine, L-proline, L-serine, glycine, L-cysteine (as L-cysteine and L-cysteine hydrochloride monohydrate) In contrast, the cytoprotective solution of the present invention is characterized by further containing L-glutamic acid, L-aspartic acid and L-tyrosine (Table 1).

  Further, regarding the concentration of amino acids and the concentration ratio of essential amino acids and non-essential amino acids, Table 2 and Table 3 are compared between the cell protection solution described in Patent Document 1 and one embodiment of the cell protection solution of the present invention. As shown in

  In particular, in the cell protection solution described in Patent Document 1 (Table 2), the concentration ratio is 1.2, whereas in one embodiment of the cell protection solution of the present invention (Table 3), the concentration ratio = 1. 4 is a feature.

  Regarding vitamins, Table 4 shows the preparation of solution (I), which is a vitamin mixture used to prepare the cell protection solution described in Patent Document 1, and one embodiment of the cell protection solution of the present invention. The comparison with the multivitamin preparation (A) which is a vitamin mixture used is shown.

  Further, regarding vitamins, Table 5 prepares one embodiment of solution (II), which is a vitamin mixture used for preparing the cell protection solution described in Patent Document 1, and the cell protection solution of the present invention. The comparison with the multivitamin preparation (B) which is a vitamin mixture used for the purpose is shown.

  Regarding vitamins, the cell protection solution described in Patent Document 1 and one embodiment of the cell protection solution of the present invention have substantially the same vitamin composition.

  For human serum albumin, glucose, sodium bicarbonate, potassium chloride, magnesium sulfate, phosphate and iron, Tables 6 and 7 show the components and their final concentrations in the cell protection solution.

  In one embodiment of the cell protection solution of the present invention disclosed so far, the sodium ion concentration is 45 mEq / L, and the osmotic pressure is a physiological saline ratio of 1.2 to 1.4. However, the osmotic pressure of 0.9% w / v physiological saline is 308 mOsm / kg.

  In one embodiment of the cell protection solution of the present invention, the multivitamins (A) and (B) are contained at concentrations of 6.6 mL / L and 50 mL / L, respectively, so that propylene glycol in the cell protection solution The concentration is 223 mM. Thereby, the osmotic pressure is set to a physiological saline ratio of 1.2 to 1.4.

  As described above, the cell protection solution of the present invention comprises 90% of cultured cells of human skeletal myoblasts over a period of at least 5 days with the cultured cells suspended at a low temperature of 2-8 ° C. The above cell viability can be maintained.

  Therefore, it is not necessary to freeze the cultured cells when storing and transporting the cultured cells, the loss of cells in the freeze-thaw process is eliminated, and operations such as removal of the frost damage protective agent after thawing are unnecessary.

The cell protection solution of the present invention is an injectable aqueous solution containing the following (a) to (j) in each amount for 1 L of the cell protection solution:
(A) Sodium bicarbonate 3.7 g; 8.4 w / v% aqueous solution 44.05 mL (44.05 mM);
(B) Potassium chloride: 302 mg; 4.05 mL (4.05 mM) of 1M aqueous solution;
(C) Calcium chloride ... 151 mg; 2.72 mL (1.36 mM) of 0.5 M aqueous solution;
(D) Dipotassium phosphate ... 0.11 g; 1.26 mL (0.63 mM) of 0.5 M aqueous solution;
(E) Magnesium sulfate: 0.2 g; 3.32 mL (1.66 mM) of 0.5 M aqueous solution;
(F) Iron: 0.000014 g; 0.7 μL of sugar-containing iron oxide containing 40 mg of iron in a 2 mL colloidal solution;
(G) Glucose ... 4.1 g;
(H) Amino acids: 10.3 mL of a comprehensive amino acid (10 w / v% amino acid aqueous solution) containing the following amino acids:
L-histidine 6.0 g / L
L-isoleucine 5.6 g / L
L-leucine 12.5g / L
L-lysine acetate 12.4 g / L
L-methionine 3.5g / L
L-Phenylalanine 9.35 g / L
L-threonine 6.5g / L
L-tryptophan 1.3g / L
L-Valine 4.5g / L
L-alanine 6.2 g / L
L-arginine 7.9 g / L
L-aspartic acid 3.8 g / L
L-cysteine 1.0 g / L
L-glutamic acid 6.5 g / L
L-proline 3.3 g / L
L-serine 2.2g / L
L-tyrosine 0.35g / L
Glycine 10.7g / L
Sodium bisulfite 0.15-0.5 g / L;
(I) Vitamins ······························· 6.6 mL of multivitamin preparation (A) containing the following vitamins and propylene glycol: 50 mL of multivitamin preparation (B):
Multivitamin preparation (A)
Ascorbic acid 20g / L
Vitamin A
(As retinol) 0.2g / L
Ergocalciferol 0.001 g / L
Thiamine hydrochloride (as thiamine) 600mg / L
Sodium riboflavin phosphate (as riboflavin) 0.72 g / L
Pyridoxine hydrochloride 0.8g / L
Nicotinamide 8g / L
Panthenol 3g / L
Propylene glycol 300g / L
Multivitamin preparation (B)
Biotin 0.012g / L
Cyanocobalamin 0.08g / L
Folic acid 0.001g / L
Propylene glycol 300 g / L;
as well as,
(J) Human serum albumin: 20 mL of 25% aqueous solution so that the final concentration is 5 g / L.

  In the above, sodium bicarbonate (final concentration 3.7 g / L) is preferably 44.05 mL of Meiron (registered trademark) 84 (Otsuka Pharmaceutical Factory), and potassium chloride (final concentration 302 mg / L). It is preferable to use 4.05 mL of KCL injection 10 mEq kit [Terumo] or KCL injection 20 mEq kit [Terumo Co., Ltd.], and calcium chloride (final concentration 151 mg / L) is calcium chloride injection 20 mEq syringe [ It is preferable to use 2.72 mL of Terumo (Terumo Corp.), and dipotassium phosphate (final concentration 0.11 g / L) is dipotassium phosphate injection 20 mEq kit [Terumo] (Terumo Corp.). 1.26 mL is preferably used, and magnesium sulfate (final concentration 0.2 g / L) is magnesium sulfate injection 20 mEq silica. It is preferable to use 3.32 mL of di [Terumo] (Terumo Co., Ltd.), and the iron (final concentration 0.000014 g / L) is Fejin (registered trademark) 40 mg (Nichiko Co., Ltd.) 0 It is particularly preferable to use 0.7 μL, and as glucose (final concentration 4.1 g / L), it is preferable to use 8.2 mL of Terumo sugar injection 50% (Terumo Co., Ltd.).

  In addition, in the above, the “total electrolyte” can be prepared by mixing (a) to (g), preferably sequentially.

  In the above, as the “total amino acid”, Variamin (registered trademark) F injection (Merck Pharmaceutical Co., Ltd.) and / or Moripron (registered trademark) F (Ajinomoto Pharma Co., Ltd.) can be used.

  In addition, in the above, “multivitamin preparation (A)” and “multivitamin preparation (B)” are neo-M. V. I. -9 Note (Irom Pharmaceutical Co., Ltd.) and M.I. V. I. -3 Note (Irom Pharmaceutical Co., Ltd.) can be used respectively.

  In order to make the total amount of the cell protecting solution a predetermined amount, it is preferable to use Japanese Pharmacopoeia water for injection, and water for injection (generic name) or distilled water for injection (generic name) can be preferably used.

  The cell protection solution prepared by the above method can be prepared by sequentially mixing water for injection and the injectable solutions (a) to (j) while maintaining the liquid temperature at 1 to 10 ° C. The thus-prepared cytoprotective solution has an osmotic pressure of 1.2 to 1.4 in physiological saline and a sodium ion concentration of about 45 mEq / L.

  The above-mentioned cell protection solution can maintain the cell viability and cell stability of cultured cells for a relatively long time in a state where skeletal myoblasts used for cell transplantation are suspended in the cell protection solution at a low temperature. Can be used to keep it high.

  Specifically, human skeletal myoblasts can be retained in a cell protection solution in a suspended state at a low temperature for at least 5 days while maintaining cell viability of 90% or more.

  Examples are shown below, but the technical scope of the present invention is not limited to the examples and should not be construed.

1. Preparation of cell protection solution (1) Preparation of total electrolyte solution While keeping the solution temperature at 1 to 10 ° C,
a) 44.05 mL (final concentration 3.7 g / L) of sodium hydrogen carbonate injection solution (8.4%) (Meiron (registered trademark) 84, Otsuka Pharmaceutical Factory);
b) 4.05 mL (final concentration 302 mg / L) of 1 molar potassium chloride solution for correction (KCL injection 10 mEq kit [Terumo], Terumo Corp.);
c) 2.72 mL (final concentration 151 mg / L) of 0.5 molar calcium chloride solution (calcium chloride injection 20 mEq syringe [Terumo], Terumo Corp.) for correction;
d) 1.26 mL (final concentration 0.11 g / L) of 0.5 mol dipotassium phosphate solution for correction (dipotassium phosphate injection 20 mEq kit [Terumo], Terumo Corporation);
e) 3.3 mol of 0.5 mol magnesium sulfate solution for correction (magnesium sulfate injection 20 mEq syringe [Terumo], Terumo Corp.) (final concentration 0.2 g / L);
f) 0.000014 g / L of sugar-containing iron oxide injection solution (Fejin (registered trademark) intravenous injection 40 mg, Nichiko Co., Ltd.) (final concentration 0.000014 g / L);
g) Glucose injection solution (50 w / v%) (Terumo sugar injection 50%, Terumo Co., Ltd.) was added to 800 mL of Japanese Pharmacopoeia water for injection to obtain a mixed solution 1.
(2) Addition of total amino acid While maintaining the liquid temperature at 1 to 10 ° C., 10.3 mL of 10% total amino acid injection solution (Varianmin (registered trademark) F injection, Merck Pharmaceutical Co., Ltd.) was added to mixture 1. It was set as the liquid mixture 2 (amino acid final concentration 1.03 g / L).
(3) Addition of multivitamin preparation While maintaining the liquid temperature at 1 to 10 ° C, mix vitamin 2 with a mixed vitamin preparation for high calorie infusion (Neo MV-9 Note, Irom Pharmaceutical Co., Ltd.). 6.6 mL and 50 mL of a mixed vitamin preparation for high calorie infusion (MV.I.-3 injection, IROM Pharmaceutical Co., Ltd.) were added to prepare a mixed solution 3.
(4) Addition of human serum albumin While maintaining the liquid temperature at 1 to 10 ° C., add 25% human serum albumin (Blood Donation Albumin 25 “Kaketsuken”, Chemistry and Serum Therapy Research Institute) to Mixture 3. In addition, mixture 4 was obtained (final concentration of human serum albumin 5 g / L).
(5) Completion of cell protection solution While maintaining the liquid temperature at 1 to 10 ° C, Japanese Pharmacopoeia water for injection was added to the mixed solution 4 to make the total volume 1 L, and the preparation of the cell protection solution was completed.

The cell protection solution prepared by the above preparation method had an osmotic pressure of about 1.3 in the physiological saline solution and a sodium ion concentration of about 45 mEq / L.
Table 8 shows changes in the osmotic pressure of the liquid during this preparation process.

  Since the osmotic pressure of the cells that have undergone culture growth is isotonic (saline solution ratio of about 1), when suspending the cells in this preparation solution, in order to avoid stress on the cells as much as possible, The suspension was mixed and stirred slowly at 10 ° C to 10 ° C.

2. Confirmation of cell stability 1) Confirmation of cell viability After preparing a cell protection solution, immediately after suspending the cells, the cells are suspended in the same manner, and then at a low temperature (2 to 8 ° C). The same amount of Trypan Blue Stain 0.4% solution (manufactured by Invitrogen) was added to and mixed with the cell suspension that had been aged for 3 days, 3 days, 4 days, and 5 days.

After mixing, 10 μL of the cell suspension was collected before the cells settled, and injected into a hemocytometer (manufactured by Elma). Immediately after the injection, the number of cells observed in the entire 9 mm ² frame was measured in ^two chambers of a hemocytometer with an inverted optical microscope (manufactured by Olympus).

  After the measurement, the average number of viable and dead cells measured from the two chambers was determined, and the ratio of the number of unstained cells to the total number of cells including stained cells was calculated.

2) Confirmation of cell purity The cell suspension immediately after preparation was centrifuged at low temperature (2 to 8 ° C) for 1 day, 3 days, 4 days, 5 days. The post-supernatant was discarded.

  To this was added 0.5% BSA-containing PBS solution to rinse the cells, and then anti-human CD56 antibody (Becton Dickinson) diluted 10-fold with 0.5% BSA-containing PBS solution was added and mixed. As a control, a negative control antibody (manufactured by Becton Dickinson) diluted 10-fold with 0.5% BSA-containing PBS solution was added and mixed.

  After mixing each antibody, it was immediately reacted in a cool and dark place for about 1 hour, and a 0.5% BSA-containing PBS solution was added to rinse the cells. Then, a 0.5% BSA-containing PBS solution was added for analysis.

  For analysis, a flow cytometer (manufactured by Becton Dickinson) was used to measure the ratio of antibody-positive cells contained in the cells mixed with each antibody. In the measurement, the positive rate of the negative control was corrected, and 5,000 to 10,000 cells were analyzed.

  After the analysis, the purity was determined from the difference in the ratio of the positive cell ratio of the cells mixed with each antibody.

3. Results and Discussion 1) Cell stability of the present invention (evaluation based on confirmation of changes in cell viability over time)
As shown in Table 9, the cell viability using the cell protection solution described in Patent Document 1 was 97% immediately after preparation, but decreased to 89% over the course of 2 days. It was. In contrast, as a result of suspending three samples of cells in the cell protection solution according to the present invention and determining the time course of cell viability, it was 94 ± 1.1% (mean ± standard deviation) immediately after preparation, 96 ± 1.3% after 1 day at 2-8 ° C. (same), 95 ± 2.0% after 3 days (same), 95 ± 1.9% after 4 days (same as above) ) And remained stable at 94 ± 2.1% (same) after 5 days.

  From the above results, it was suggested that the cell viability of the present invention is more stable.

  About this result, the transition (average value) of the cell viability when using the cell protection solution of the present invention in FIG. 1 and the case of using the cell protection solution described in Patent Document 1 as a comparative control are shown in FIG. The transition of cell viability (document description value) is shown.

2) Cell stability of the present invention (supported by confirming changes in cell purity over time)
In order to confirm that the results of cell viability obtained above surely reflect the viability of skeletal myoblasts, the purity of skeletal myoblasts contained in the cell suspension of the present invention was determined over time. It was done by confirming.

  For confirmation, the time course of cell purity was determined for the prepared cell suspensions of 3 specimens.

  As a result, the cell purity was 95 ± 2.3% (mean ± standard deviation) immediately after preparation, 94 ± 1.9% (same) for 1 day at low temperature (2 to 8 ° C.), and 94 ± 9 for 3 days. 1.9% (same as above), 94 days 1.9% (same) over 4 days, and 93 ± 2.4% (same) over 5 days, indicating stable.

  As a result of the above, it became clear that the cells that showed stable viability over time were surely skeletal myoblasts, and the cell viability of the present invention with respect to the cell protection solution described in Patent Document 1 was improved. The superiority of the cell protection solution was confirmed.

  FIG. 2 shows the time course of cell purity (average value. Error bars are maximum and minimum values).

  An object of the present invention is to perform cell storage / transport without freezing at low temperatures. Therefore, the present inventors conducted a keen study on conditions for stably maintaining cell viability over a longer period of time with respect to the cell protection solution composition described in Patent Document 1.

As a result of the examination, it was found that the cell viability was stably maintained without swelling for 5 days with time in a low temperature (2 to 8 ° C.) environment after cell suspension under the following conditions. :
a) Concerning the total amino acids constituting the cell protection solution, L-glutamic acid, L-aspartic acid, L-tyrosine were included;
b) The multivitamin preparation constituting the cell protection solution contains propylene glycol for hypertonicization of the solution intended to suppress cell swelling under low temperature; and
c) For the total electrolyte solution constituting the cell protection solution, the sodium ion concentration was set to 45 mEq / L in order to further stabilize the cell viability under the low temperature hypertonic solution.

  Therefore, there is no worry about the loss at the time of cell thawing caused by freezing that is normally performed in the storage and transport of cells, and the storage and transport of cells that do not require operations such as washing and removal of the freezing protection agent is stable and long-lasting. It was confirmed that it could be done.

  After culturing and proliferating skeletal myoblasts separated from skeletal muscle biopsied in a medical institution, the cells can be packaged in a state of maintaining activity and provided to the medical institution.

The cell viability (mean value) (■) when the cell protection solution of the present invention is used and the cell viability (document value) (♦) when the cell protection solution described in Patent Document 1 is used. Show. The time-dependent transition (average value) of the cell purity at the time of using the cell protection solution according to the present invention is shown.

Claims (19)

  Amino acids, vitamins, electrolytes, iron, sugar and serum albumin, the amino acids have a final concentration of 0.5 to 1.5 g / L, and the concentration ratio of essential amino acids to non-essential amino acids is 1.3 to A solution for cell protection that is 1.7 and stably holds animal cells for a long time at low temperature without freezing.   The liquid for cell protection according to claim 1, wherein the amino acid contains L-glutamic acid, L-aspartic acid and L-tyrosine.   The cell protection solution according to claim 1 or 2, wherein the vitamin preparation has a final concentration of all vitamins in the cell protection solution of 0.02 to 0.3 g / L.   The said electrolyte is the liquid for cell protection in any one of Claims 1-3 which sets the final concentration of the sodium ion in the said liquid for cell protection to 40-50 mEq / L.   The amino acid is L-isoleucine, L-leucine, L-lysine acetate, L-methionine, L-phenylalanine, L-threonine, L-tryptophan, L-valine, L-alanine, L-arginine, L-histidine, L The cell protection solution according to any one of claims 1 to 4, comprising -proline, L-serine, L-glycine, L-cysteine, L-glutamic acid, L-aspartic acid and L-tyrosine.   The amino acids were L-isoleucine 5.6 g / L, L-leucine 12.5 g / L, L-lysine acetate 12.4 g / L, L-methionine 3.5 g / L, L-phenylalanine 9 .35 g / L, L-threonine 6.5 g / L, L-tryptophan 1.3 g / L, L-valine 4.5 g / L, L-alanine 6.2 g / L, L-arginine 7 .9 g / L, L-histidine 6.0 g / L, L-proline 3.3 g / L, L-serine 2.2 g / L, glycine 10.7 g / L, L-cysteine 1.0 g / L, an aqueous solution containing L-glutamic acid at a concentration of 6.5 g / L, L-aspartic acid at a concentration of 3.8 g / L, and L-tyrosine at a concentration of 0.35 g / L, respectively. The cell protection solution according to 1.   The said vitamin preparation contains ascorbic acid, vitamin A, ergocalciferol, thiamine hydrochloride, riboflavin sodium phosphate, pyridoxine hydrochloride, nicotinamide, panthenol, tocopherol acetate, biotin, folic acid, and cyanocobalamin. The cell protection solution according to any one of the above.   The liquid for cell protection according to any one of claims 1 to 7, wherein the vitamin preparation further contains propylene glycol.   The vitamin preparation is 20 g / L ascorbic acid, 0.2 g / L vitamin A is retinol, 0.001 g / L ergocalciferol, thiamine hydrochloride is 600 mg / L thiamin, and riboflavin sodium phosphate is 0 riboflavin. .72 g / L, pyridoxine hydrochloride 0.8 g / L, nicotinamide 8 g / L, panthenol 3 g / L, tocopherol acetate 2 g / L, biotin 0.012 g / L, folic acid 0.08 g / L The cell protection solution according to any one of claims 1 to 8, which is an aqueous solution containing 0.001 g / L of cyanocobalamin and 300 g / L of propylene glycol.   The cell protection solution according to any one of claims 1 to 9, wherein the electrolyte contains sodium hydrogen carbonate, potassium chloride, magnesium sulfate, and dipotassium phosphate.   In the cell protection solution, the electrolyte was 3.7 g / L sodium bicarbonate, 302 mg / L potassium chloride, 0.2 g / L magnesium sulfate, and 0.11 g / L dipotassium phosphate. The cell protection solution according to any one of claims 1 to 10, which is contained at a final concentration.   The cell protection solution according to claim 1, wherein the iron is provided by sugar-containing iron oxide.   The cell protection solution according to any one of claims 1 to 12, wherein the iron is contained in the cell protection solution at a final concentration of 0.000014 g / L.   The cell protection solution according to any one of claims 1 to 13, wherein the sugar is glucose.   The cell protection solution according to any one of claims 1 to 14, wherein the sugar is contained in the cell protection solution at a final concentration of 4.1 g / L.   The liquid for cell protection according to any one of claims 1 to 15, wherein the serum albumin is contained in the cell protection liquid at a final concentration of 0.1 to 10 g / L.   The cell protection solution according to any one of claims 1 to 16, wherein the serum albumin is contained in the cell protection solution at a final concentration of 5 g / L.   The solution for cell protection according to any one of claims 1 to 17, wherein the osmotic pressure is 1.2 to 1.4 times that of physiological saline. A method for producing a cell-protecting solution by mixing a total electrolyte solution, a total amino acid, a total vitamin preparation (A) and (B), and human serum albumin with water, each element comprising the following (1) to (5) A method for producing a cell-protecting solution, characterized by comprising:
(1) Comprehensive electrolyte solution: 44.05 mL of 8.4 w / v% aqueous sodium hydrogen carbonate solution for 1 L of cell protection solution; 4.05 mL of 1 M potassium chloride aqueous solution and 2. 5 M calcium chloride aqueous solution 2. 72mL, 0.5M dipotassium phosphate aqueous solution 1.26mL, 0.5M magnesium sulfate aqueous solution 3.32mL, iron containing 40mg in 2mL colloidal solution 0.7μL of sugar-containing iron oxide, 50w / v glucose 8.2 mL of aqueous solution;
(2) Total amino acids: L-histidine 6.0 g / L, L-isoleucine 5.6 g / L, L-leucine 12.5 g / L, L-lysine acetate 12.4 g / L, L-methionine 3.5 g / L, L-phenylalanine 9.35 g / L, L-threonine 6.5 g / L, L-tryptophan 1.3 g / L, L-valine 4.5 g / L, L-alanine 6.2 g / L, L-arginine 7.9 g / L, L-aspartic acid 3.8 g / L, L-cysteine 1.0 g / L, L-glutamic acid 6.5 g / L, L- An aqueous solution containing 3.3 g / L of proline, 2.2 g / L of L-serine, 0.35 g / L of L-tyrosine and 10.7 g / L of glycine was used for 1 L of cell protection solution. 10.3 mL;
(3) Multivitamin preparation (A): 20 g / L of ascorbic acid, 0.2 g / L of vitamin A as retinol, 0.001 g / L of ergocalciferol, 600 mg / L of thiamine hydrochloride as thiamine, riboflavin phosphate Cell protection of aqueous solutions containing 0.72 g / L sodium riboflavin, 0.8 g / L pyridoxine hydrochloride, 8 g / L nicotinamide, 3 g / L panthenol and 300 g / L propylene glycol. 6.6 mL for 1 L of working solution;
(4) Multivitamin preparation (B): Cell protection for aqueous solutions containing biotin at 0.012 g / L, cyanocobalamin at 0.08 g / L, folic acid at 0.001 g / L and propylene glycol at 300 g / L. 50 mL for 1 L of working solution; and
(5) Human serum albumin: 20 mL of 25% human serum albumin aqueous solution for 1 L of cell protection solution.

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